Under pathological conditions of acute and chronic forms of neurodegeneration overactivation of NMDA receptors is a key event for triggering neuronal cell death. NMDA receptors are composed of members of two subunit families, namely NR-1 (8 different splice variants) and NR-2 (A to D) originating from different genes. Members from the two subunit families show a distinct distribution in different brain areas. Heteromeric combinations of NR-1 members with different NR-2 subunits result in NMDA receptors displaying different pharmaceutical properties. Possible therapeutic indications for NMDA receptor subtype specific blockers include acute forms of neurodegeneration caused, e.g., by stroke and brain trauma, and chronic forms of neurodegeneration such as Alzheimer,s disease, Parkinson,s disease, Hantington,s disease, ALS (amyotrophic lateral sclerosis) and neurodegeneration associated with bacterial or viral infections.
The present invention relates to novel 4-hydroxy-piperidine derivatives, a process for their manufacture, and NMDA receptor subtype specific blocker compositions containing such 4-hydroxy-piperidine derivatives.
The present invention relates to 4-hydroxy-piperidine derivatives, exhibiting NMDA receptor subtype specific blocker activity.
The novel 4-hydroxy-piperidine derivatives of the present invention have the general formula (I), 
wherein
X denotes xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94CH2xe2x80x94, xe2x80x94CHxe2x95x90, xe2x80x94CHOHxe2x80x94, xe2x80x94COxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94 or xe2x80x94SO2xe2x80x94;
R1-R4 are, independently from each other, hydrogen, hydroxy, lower-alkyl-sulfonylamino, 1- or 2-imidazolyl or acetamido;
R5-R8 are, independently from each other, hydrogen, hydroxy, lower-alkyl, halogen, lower-alkoxy, trifluoromethyl or trifluoromethyloxy;
a and b may be a double bond, provided that when xe2x80x9caxe2x80x9d is a double bond, xe2x80x9cbxe2x80x9d cannot be a double bond;
n is 0-2;
m is 1-3;
p is 0 or 1
and to pharmaceutically acceptable addition salts thereof.
The compounds of formula I and their salts are distinguished by valuable therapeutic properties. Compounds of the present invention are NMDA(N-methyl-D-aspartate)-receptor subtype selective blockers, which have a key function in modulating neuronal activity and plasticity which makes them key players in mediating processes underlying development of the CNS including learning and memory formation and function.
The objects of the invention are the compounds of formula I and pharmaceutically acceptable addition salts thereof, racemic mixtures and their corresponding enantiomers and the preparation of the above-mentioned compounds. Further objects of the present invention include medicaments containing the compounds and their manufacture as well as the treatment and control of illness using the compounds and medicaments set forth herein.
The following definitions of the general terms are used the present specification and apply irrespective of whether the terms appear alone or in combination.
As used herein, the term xe2x80x9clower alkylxe2x80x9d denotes a straight or branched-chain alkyl group containing from 1 to 4 carbon atoms, for example, methyl, ethyl, propyl, isopropyl, n-butyl, i-butyl, 2-butyl and t-butyl.
The term xe2x80x9chalogenxe2x80x9d denotes chlorine, iodine, fluorine and bromine.
The term xe2x80x9clower alkoxyxe2x80x9d denotes a group wherein the alkyl residue is as defined above.
The term xe2x80x9cleaving groupxe2x80x9d has the meaning conventionally used, and refers to, for example, halogen, alkylsulfonyloxy, arylsulfonyloxy and the like. The most preferred leaving group in the present case is a halogen.
The term xe2x80x9cpharmaceutically acceptable addition saltsxe2x80x9d embraces salts with inorganic and organic acids generally known to a person skilled in the art, such as hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, citric acid, formic acid, fumaric acid, maleic acid, acetic acid, succinic acid, tartaric acid, methane-sulfonic acid, p-toluenesulfonic acid and the like.
Compounds of formula I wherein X is xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94CHOH or xe2x80x94CH2xe2x80x94 are preferred.
Exemplary preferred compounds in which X denotes xe2x80x94Oxe2x80x94, are:
(RS)-1-(5-hydroxy-2,3-dihydro-benzofuran-2-ylmethyl)-4-(4-methyl-benzyl)-piperidine-4-ol,
(RS)-4-benzyl-1-(5-hydroxy-2,3-dihydro-benzofuran-2-ylmethyl)-piperidine-4-ol,
(RS)-4-(4-fluoro-benzyl)-1-(5-hydroxy-2,3-dihydro-benzofuran-2-ylmethyl)-piperidine-4-ol,
(RS)-4-(4-ethyl-benzyl)-1-(5-hydroxy-2,3-dihydro-benzofuran-2-ylmethyl)-piperidine-4-ol,
(S)-1-(5-hydroxy-2,3-dihydro-benzofuran-2-ylmethyl)-4-(4-methyl-benzyl)-piperidine-4-ol,
(S)-1-(5-hydroxy-2,3-dihydro-benzofuran-2-ylmethyl)-4-(4-chloro-benzyl)-piperidine-4-ol,
(RS)-N-[2-{4-hydroxy-4-(4-methyl-benzyl)-piperidine-1-ylmethyl}-2,3-dihydrobenzofuran-5-yl]-methane sulfonamide; and
(RS)-N-[2-{4-hydroxy-4-(4-methyl-benzyl)-piperidine-1-ylmethyl}-2,3-dihydrobenzofuran-5-yl]-methane sulfonamide.
Exemplary preferred compounds in which X denotes xe2x80x94CHOHxe2x80x94 are:
(1RS,2RS) and (1RS,2SR)-2-[4-hydroxy-4-(4-methyl-benzyl)-piperidine-1-ylmethyl]-indan-1,5-diol,
(1RS,2RS)-1-(1,6-dihydroxy-1,2,3,4-tetrahydro-naphthalen-2-ylmethyl)-4-(4-methyl-benzyl)-piperidine-4-ol; and
(1RS,2RS)-2-(4-benzyl-4-hydroxy-piperidine-1-yl-methyl)-6-hydroxy-1,2,3,4-tetrahydronaphthalen-1-ol.
Exemplary preferred compounds in which X denotes xe2x80x94CH2xe2x80x94 are:
(RS)-1-(5-hydroxy-indan-2-ylmethyl)-4-(4-methyl-benzyl)-piperidine-4-ol; and
(RS)-1-(6-hydroxy-1,2,3,4-tetrahydro-naphthalen-2-ylmethyl)-4-(4-methyl-benzyl)-piperidine-4-ol.
Another exemplary preferred compound in which X denotes xe2x80x94NHxe2x80x94 is:
(RS)-2-[4-hydroxy-4-(4-methyl-benzyl)-piperidine-1-ylmethyl]-2,3-dihydro-1H-indol-5-ol.
The present compounds of formula I and their pharmaceutically acceptable salts can be prepared by methods known in the art, for example, by the processes (a-j) described below:
a) reacting a compound of the formula (II) 
wherein R1-R4, X, a, b, n and m have the meaning given in general formula I and L is OH or a leaving group, for example, halogen or xe2x80x94Oxe2x80x94tosyl,
with a compound of the formula (III) 
wherein R5-R8 and p have the meaning given in general formula I or
b) reacting a compound of the formula (IV) 
wherein the substituents have the meaning given in general formula I,
with a compound of the formula (IIIA) 
in the presence of paraformaldehyde to give a compound of the formula (IA) 
wherein m is 1 and the other substituents have the meaning given in general formula I, or
c) dehydrating a compound of the formula (IB) 
to give a compound of the formula (IC) 
wherein m is 1 and the other substituents have the meaning given in general formula I, or
d) reducing a compound of the formula IA to give a compound of the formula IB, or
e) debenzylating a compound of the formula (V) 
or
f) reacting a compound of formula I, wherein one of R1-R4 is an amino group with a lower-alkyl-sulfonyl halogen to give a compound of formula I, wherein one of R1-R4 is a lower-alkyl-sulfonyl-amino group, or
g) hydrogenating the isolated double bond in a compound of formula I, or
h) cleaving off (a) hydroxy or amino protecting group(s) present as (a) substituent(s) R1-R4 or as Xxe2x80x2=xe2x80x94N(protecting group)-, or
i) oxidizing a compound of formula I, wherein X represents xe2x80x94Sxe2x80x94 or xe2x80x94SOxe2x80x94 to yield the corresponding sulfonyl (xe2x80x94SO2) compound; and
j) if desired, converting the compound of formula I obtained into a pharmaceutically acceptable addition salt.
In accordance with process variant a), a mixture of a compound of formula II and of formula III, wherein the leaving group L in formula II is, for example, bromine, was dissolved in a suitable solvent, for example in DMF and heated to about 80-90xc2x0 C. This reaction was carried out in the presence of a base, preferably triethylamine. The compound of formula I is then separated in conventional manner. When one of R1-R4 in formula II is a hydroxy group these groups are protected by groups conventionally used.
Examples of such groups are described in Green, T., Protective Groups in Organic Synthesis, Chapter 7, John Wiley and Sons, Inc. (1981), pp 218-287. Most preferred are the benzyloxy or alkyloxy groups. This reaction can be carried out by known methods, for example by hydrogenation with Pd/C (10%) or borontribromide-dichloromethane solution.
Process variant b) describes a process to obtain compounds of formula I wherein X is a xe2x80x94COxe2x80x94 group.
A compound of the formula IV is heated in a suitable solvent, for example in DMF together with a compound of formula IIIA in the presence of paraformaldehyde. This reaction is carried out at about 80xc2x0 C. in conventional manner.
In accordance with process variant c), a compound of formula IB can be dehydrated in the presence of ethanolic HCl in conventional manner. Compounds of formula I in which xe2x80x9caxe2x80x9d represents a double bond are obtained.
Variant d) describes a method for reducing of compounds of formula IA to give compounds of the formula IB. This reaction is carried out in conventional manner, preferably the reaction is carried out in the presence of LiAIH4 in THF and at temperature of about 5-10xc2x0 C.
In accordance with process variant e), a compound of formula I is obtained, wherein one of R1-R4 is hydroxy. This process is carried out by debenzylating a compound of formula V, provided that none of R5-R8 is halogen. The debenzylation is carried out in conventional manner. For example, a compound of formula V is dissolved in a suitable solvent or mixture of solvents such as ethanol and ethylacetate, and hydrogenated in the presence of Pd on C at room temperature and atmospheric pressure.
In accordance with process variant f), a compound of formula I can be obtained, wherein one of R1-R4 is a lower-alkyl-sulfonyl-amino group. This reaction is carried out by treating a compound of formula I, wherein one of R1-R4 is an amino group, with a lower-alkyl-sulfonylhalogen, such as methane sulfonylchloride, in a suitable solvent, such as methylene chloride, in the presence of pyridine at room temperature.
The hydrogenation of a compound of formula I, wherein one of xe2x80x9caxe2x80x9d or xe2x80x9cbxe2x80x9d is a double bond in accordance with process variant g) is carried out in conventional manner, for example in the presence of Pd/C in ethylacetate under hydrogen atmosphere for about 24 hours at room temperature. Protecting groups, for example the hydroxy group, can be cleaved off by methods described above. Suitable protecting groups and methods for their cleavage will be familiar to any person skilled in the art; although of course there can be used only those protecting groups which can be cleaved off by methods under conditions of which other structural elements in the compounds are not affected.
The oxidation of compounds of formula I, wherein X is xe2x80x94Sxe2x80x94 or xe2x80x94SOxe2x80x94, is carried out in conventional manner. In accordance with process variant i), a compound of formula I, wherein X represents xe2x80x94Sxe2x80x94 or xe2x80x94SOxe2x80x94, is oxidized to yield the corresponding sulfonyl (SO2xe2x80x94) compound. The oxidation can be carried out in the presence of Oxone(copyright) (potassium monopersulfate triple salt) at room temperature or in the presence of metachloroperbenzoic acid.
The addition salts of the compounds of formula I are especially well suited for pharmaceutical use.
As mentioned earlier, the compounds of formula I and their pharmaceutically usable addition salts possess valuable pharmacodynamic properties. They are NMDA-receptor subtype selective blockers, which have a key function in modulating neuronal activity and plasticity which makes them key players in mediating processes underlying development of CNS as well as learning and memory formation.
The compounds were investigated in accordance with the tests given hereinafter.
Male Fxc3xcillinsdorf albino rats weighing between 150-200 g were used. Membranes were prepared by homogenization of the whole brain minus cerebellum and medulla oblongata with a Polytron (10.000 rpm, 30 seconds), in 25 volumes of a cold Tris-HCl 50 mM, EDTA 10 mM, pH 7.1 buffer. The homogenate was centrifuged at 48.000 g for 10 minutes at 4xc2x0 C. The pellet was resuspended using the Polytron in the same volume of buffer and the homogenate was incubated at 37xc2x0 C. for 10 minutes. After centrifugation the pellet was homogenized in the same buffer and frozen at xe2x88x9280xc2x0 C. for at least 16 hours but not more than 10 days. For the binding assay the homogenate was thawed at 37xc2x0 C., centrifuged and the pellet was washed three times as above in a Tris-HCl 5 mM, pH 7.4 cold buffer. The final pellet was resuspended in the same buffer and used at a final concentration of 200 xcexcg of protein/ml. 3H-Ro 25-6981 binding experiments were performed using a Tris-HCl 50 mM, pH 7.4 buffer. For displacement experiments 5 nM of 3H-Ro 25-6981 were used and non specific binding was measured using 10 xcexcM of tetrahydroisoquinoline and usually it accounts for 10% of the total. The incubation time was 2 hours at 4xc2x0 C. and the assay was stopped by filtration on Whatmann GF/B glass fiber filters (Unifilter-96, Packard, Zxc3xctrich, Switzerland). The filters were washed 5 times with cold buffer. The radioactivity on the filter was counted on a Packard Top-count microplate scintillation counter after addition of 40 mL of microscint 40 (Canberra Packard S. A., Zxc3xcrich, Switzerland).
The effects of compounds were measured using a minimum of 8 concentrations and repeated at least once. The pooled normalized values were analyzed using a non-linear regression calculation program which provide IC50 with their relative upper and lower 95% confidence limits (RS1, BBN, USA).
Male Fxc3xcllinsdorf albino rats weighing between 150-200 g were used. Membranes were prepared by homogenization of the whole brain minus cerebellum and medulla oblongata with a Plytron (10.000 rpm, 30 seconds), in 25 volumes of a cold Tris-HCl 50 mM, EDTA 10 mM, pH 7.1 buffer. The homogenate was centrifuged at 48.000 g for 10 minutes at 4xc2x0 C. The pellet was resuspended using the Polytron in the same volume of buffer and the homogenate was incubated at 37xc2x0 C. for 10 minutes. After centrifugation the pellet was homogenized in the same buffer and frozen at xe2x88x9280xc2x0 C. for at least 16 hours but not more than 10 days. For the binding assay the homogenate was thawed at 37xc2x0 C., centrifuged and the pellet was washed three times as above in a Tris-HCl 5 mM, pH 7.4 cold buffer. The final pellet was resuspended in the same buffer and used at a final concentration of 200 xcexcg of protein/ml.
3H-Prazosine binding experiments were performed using a Tris-HCl 50 mM, pH 7.4 buffer. For displacement experiments 0.2 nM of 3H-Prazosine were used and non specific binding was measured using 100 xcexcM of Chlorpromazine. The incubation time was 30 minutes at room temperature and the assay was stopped by filtration on Whatman GF/B glass fiber filters (Unifilter-96, Canberra Packard S. A., Zxc3xcrich, Switzerland). The filters were washed 5 times with cold buffer. The radioactivity on the filter was counted on a Packard Top-count microplate scintillation counter after addition of 40 ml of microscint 40 (Canberra Packard S. A., Zxc3xcrich, Switzerland). The effects of compounds were measured using a minimum of 8 concentrations and repeated at least once. The pooled normalized values were analyzed using a non-linear regression calculation program which provide IC50 with their relative upper and lower 95% confidence limits (RS1, BBN, USA).
cDNA clones coding for the subunits NR1C and NR2A of the NMDA receptor (see Hollmann and Heinemann, 1994, Annu. Rev. Neurosci. 17:31 for nomenclature of NMDA receptor subunits) were isolated from a rat brain xcexgt11 cDNA library as published elsewhere (Sigel et al., 1994, J. Biol. Chem. 269:8204). The clone for the subunit NR2B of the rat brain NMDA receptor was obtained from S. Nakanishi (Kyoto, Japan). The cDNAs encoding rat NR1C, NR2A and NR2B were subcloned into the expression vector pBC/CMV (Bertocci et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88:1416), placing transcription of the cDNA under control of the human cytomegalovirus promoter. CsCl-purified expression plasmids were mixed in a 1:3 ratio of NR1C:NR2A or NR1C:NR2B in injection buffer (88 mM NaCl, 1 mM KCl, 15 mM HEPES, at pH 7.0). Oocytes of South African frogs (Xenopus laevis) were used for expressing either a combination of the NR1C and NR2A subunits or the NR1C and NR2B subunits. 12 to 120 pg of a 1:3 (NR1C:NR2B) mixture of the respective cDNA species were injected into the nucleus of every oocyte. On the following two days the ion current through the NMDA receptor channels was measured in voltage clamp experiments for the methods of cDNA expression in oocytes and voltage-clamping (see, e.g., Bertrand et al., 1991, Methods in Neurosciences 4:174). The membrane potential was clamped to xe2x88x9280 mV and the receptors were activated by applying a modified Ringer,s solution containing the NMDA-receptor agonists L-glutamate (Glu) and glycine (Gly). Different agonist concentrations were chosen for either subunit combination to account for the different agonist sensitivities of the two types of receptors (2.7 xcexcM Glu plus 0.9 xcexcM Gly for NR1C+NR2A and 1.3 xcexcM Glu plus 0.07 xcexcM Gly for NR1C+NR2B). The agonists were applied for 15 s intervals once every 2.5 min by rapid superfusion of the oocyte with agonist containing solution and the amplitude of the agonist-evoked current was measured immediately before the end of each application. After a series of initial control applications the antagonist to be tested was added to both, the basal Ringer,s and the agonist containing solution. The antagonist concentration applied to oocytes expressing the NR2A subunit was 10 xcexcmol/l, whereas 0.1 xcexctmol/ were applied to the NR2B expressing oocytes. Four to six oocytes were tested for every compound and NMDA receptor subtype. Oocytes were exposed to the compounds for 5 to 30 min depending on the time needed for reaching an equilibrium block of the NMDA receptor current. For every oocyte the decrease of the current amplitude was expressed as a percentage of the control current measured before application of the compound. Figures in the table are arithmetic mean values of these percentage values.
The thus-determined activity of some compounds in accordance with the invention will be evident from the following table 1.
(01) 1-(6-Hydroxy-3,4-dihydro-naphthalene-2-ylmethyl)-4-(4-methyl-benzyl)-piperidine-4-ol
(02) (RS)-1-(5-Hydroxy-2,3-dihydro-benzofuran-2-ylmethyl)-4-(4-methyl-benzyl)-piperidin-4-ol hydrochloride
(04) (RS)-4-Benzyl-1-(5-hydroxy-2,3-dihydro-benzofuran-2-ylmethyl)-piperidin-4-ol hydrochloide
(05) (RS)-4-(4-fluoro-benzyl)-1-(5-hydroxy-2,3-dihydro-benzofuran-2-ylmethyl)-piperidin-4-ol hydrochloride
(07) (RS)-4-(4-ethyl-benzyl)-1-(5-hydroxy-2,3-dihydro-benzofuran-2-ylmethyl)-piperidin-4-ol hydrochloride
(13) (S)-1-(5-Hydroxy-2,3-dihydro-benzofuran-2-ylmethyl)-4-(4-methyl-benzyl)-piperidin-4-ol hydrochloride
(14) (S)-1-(5-Hydroxy-2,3-dihydro-benzofuran-2-ylmethyl)-4-(4-chloro-benzyl)-piperidin-4-ol hydrochloride
(16) (1RS,2RS) and (IRS, 2SR)-2-[4-Hydroxy-4-(4-methyl-benzyl)-piperidine-1-ylmethyl]-indan-1,5-diol fumarate salt
(19) (RS)-1-(5-Hydroxy-indan-2-ylmethyl)-4-(4-methyl-benzyl)-piperidin-4-ol
(21) (RS)-2-[4-Hydroxy-4-(4-methyl-benzyl)-piperidin-1-ylmethyl]-2,3-dihydro-1H-indol-5-ol
(22) (RS)-N(2-[4-Hydroxy-4-{4-methyl-benzyl}-piperidin-1-ylmethyl]-2,3-dihydrobenzofuran-5-yl)-methane sulfonamide hydrochloride
(23) (RS)-N(2-[4-hydroxy-4-(4-methyl-benzyl)-piperidin-1-ylmethyl]-2,3-dihydrobenzofuran-5-yl)-methane sulfonamide hydrochloride
(27) (1RS, 2RS)-1-(1,6-Dihydroxy-1,2,3,4-tetrahydro-naphthalen-2-ylmethyl)-4-(4-methyl-benzyl)-piperidin-4-ol fumarate salt
(29) (1RS,2RS)-2-(4-benzyl-4-hydroxy-piperidin-1-yl-methyl)-6-hydroxy-1,2,3,4-tetrahydro-naphthalen-1-ol
(30) (RS)-1-(6-Hydroxy-1,2,3,4-tetrahydro-naphthalen-2-ylmethyl)-4-(4-methyl-benzyl)-piperidin-4-ol hydrochloride
By screening, compounds of formula I could be identified as NMDA receptor subtype selective blockers andxe2x80x94for selected compoundsxe2x80x94the preference for NMDAR-2B subunits could be demonstrated by eletrophysiological characterization using cloned NMDA receptor subtypes expressed oocytes.
The compounds of formula I and their salts, as herein described, can be incorporated into standard pharmaceutical dosage forms, for example, for oral or parenteral application with the usual pharmaceutical adjuvant materials, for example, organic or inorganic inert carrier materials, such as, water, gelatin, lactose, starch, magnesium stearate, talc, vegetable oils, gums, polyalkylene-glycols and the like. The pharmaceutical preparations can be employed in a solid form, for example, as tablets, suppositories, capsules, or in liquid form, for example, as solutions, suspensions or emulsions. Pharmaceutical adjuvant materials can be added and include preservatives stabilizers, wetting or emulsifying agents, salts to change the osmotic pressure or to act as buffers. The pharmaceutical preparations can also contain other therapeutically active substances.
The compounds of this invention may be administered in amounts that are pharmaceutically effective for the treatment of various acute forms of neurodegeneration. Particularly possible therapeutic indications for the NMDA receptor subtype specific blockers of this invention include chronic forms of neurodegeneration such as Alzheimer""s disease, Parkinson""s disease, Huntington""s disease, and ALS (amyotrophic lateral sclerosis). Also treatable by the administration of pharmaceutically effective amounts of the NMDA receptor subtype specific blockers of this invention are acute forms of neurodegeneration caused by stroke and brain trauma, as well as neurodegeneration associated with bacterial or viral infections.
The daily dose of compounds of formula I to be administered varies with the particular compound employed, the chosen route of administration and the recipient. Representative of a method for administering the compounds of formula I is by the oral and parenteral type administration route. An oral formulation of a compound of formula I is preferably administered to an adult at a dose in the range of 150 mg to 1.5 g per day. A parenteral formulation of a compound of formula I is preferably administered to an adult at a dose in the range of 5 to 500 mg per day.